Isotope Ratio Mass Spectrometry (IRMS) is a technique that finds application across many fields including geosciences, archaeology, medicine, geology, biology, food authenticity and forensic science. Accurate and precise measurement of variations in the abundances of isotopic ratios of light elements in a sample such as 13C/12C (δ13C), 15N/14N (δ15N), 18O/16O (δ18O), D/H, and 34S/32S (δ34S), relative to an isotopic standard, can provide information on the geographical, chemical and biological origins of substances, allowing differentiation between samples that are otherwise chemically identical. The δ values are defined in a specific way. For example, δ13C is defined as:
            δ      13        ⁢          C      ⁡              (                  0          00                )              =            (                                                  (                                                                                                                                  13                                    ⁢                  C                                                                                           12                                    ⁢                  C                                            )                        sample                                              (                                                                                                                                  13                                    ⁢                  C                                                                                           12                                    ⁢                  C                                            )                                      isotopic              ⁢                                                          ⁢              standard                                      -        1            )        *    1000  
A typical EA-IRMS instrument is formed of six main sections: a sample introduction system, a sample preparation system, an electron ionisation source, a magnetic sector analyser, a Faraday collector detector array, and a computer controlled data acquisition system. The sample is split into atoms/molecules and/or compounds by the sample preparation system. The electron ionisation source ionizes the prepared sample and the resulting sample ions are spatially separated in the magnetic sector analyser. The Faraday collector comprises a detector array which detects the spatially separated ions, and the computer controlled data acquisition system generates mass spectra from the Faraday collector outputs.
Sample preparation may be achieved in a number of different ways, with advantages and disadvantages to each. The two best-known groups of techniques for sample preparation are those which carry out elemental analysis for the whole sample (EA-IRMS), and those which first separate the chemical substances of the sample by gas chromatography before splitting the separated substances into atoms/molecules and/or compounds (GC-IRMS). Liquid chromatography (LC-IRMS) has also been explored for sample preparation but is less commonly used.
EA-IRMS is a measurement technique which analyses the whole sample at the same time, to investigate the variations in the abundances of isotope ratios in the whole sample. FIG. 1a shows a highly schematic arrangement of the sample introduction and preparation part (see above) of an EA-IRMS system. The system is under the control of a system controller 100 as may be seen.
A sample (not shown in FIG. 1a) is weighed and placed in a combustible capsule (also not shown in FIG. 1a). The combustible capsule is sealed with the sample inside and is usually made of tin, although aluminium or silver may be used instead.
An autosampler carousel 10 is positioned above a combustion furnace 20. Helium purge gas is supplied to the autosampler 10, typically at a rate of 20-300 ml/min, by a first gas supply control 14 from a first Helium bottle 13 to reduce air intake. The He purge gas flows out of the sampler via the outlet pipe 18. The autosampler carousel 10 injects the sealed sample capsule into the combustion furnace 20 in a carrier gas flow of helium supplied by a second gas supply control 15 from a second Helium bottle 16. The sample is combusted in the combustion furnace 20, under the control of the system controller 1. Pulsed oxygen may optionally be employed to aid combustion. The oxygen is supplied from an oxygen bottle 17, also under the control of the second gas supply control 15.
The sample matrix breaks down into its constituent elemental components (mostly atoms) and is conveyed by the carrier gas flow of Helium from the second Helium bottle 16, across an oxygen donor compound such as Cr2O3, WO3, or CuO. The oxygen donor is present to ensure complete oxidation of the elemental components, particularly of carbon, nitrogen and sulfur evolved from the sample matrix. Typically the reactor zone (containing the oxygen donor) in the combustion furnace 20 is held at a temperature of between 400 and 1100 degrees Celsius, with an ideal range of between 900 and 1050 degrees Celsius. The Helium carrier gas employs a maximum flow rate of up to 1000 mL/min, but typically in the range of 40 to 200 mL/min.
The resulting products may be one or more of NOx, CO2, SO2 and/or H2O. After the oxidation a reduction takes place. For example, to measure δ15N, NOx has to be reduced to N2. This may be carried out either using separate, serially arranged combustion and reduction furnaces (as shown in FIG. 1a), or alternatively by combining both into a single reactor heated by the same furnace.
In particular, the arrangement shown in FIG. 1a, employs a separate reduction oven 30, arranged downstream of the combustion furnace 20 and heated separately to the combustion furnace 20. In the arrangement of FIG. 1a, the sample is generally swept across the oxygen donor material in the reactor zone of the combustion furnace 20 using the Helium carrier gas, and then transferred to the reduction oven 30, via a stainless steel/sulfinert capillary or heated bridge, which contains metallic copper (not shown in FIG. 1a). The reduction oven 30 is generally held at a temperature between 450-900° C. and is designed to reduce NOx and NO gas species (for example) to N2, reduce SO3 to SO2 and absorb excess O2 not used in the combustion reaction.
In the alternative arrangement, where the combustion and reduction processes may instead be combined in the same reactor, heated by the same furnace, the analyte gases first pass across the oxygen donor compound. The gases are then conveyed onward to metallic copper within the same reactor. Here, they undergo the same chemical reaction as described above in respect of the serially arranged furnaces illustrated in FIG. 1a. 
In either case (separate or combined combustion and reduction furnaces/ovens), the resultant gases are then directed through a moisture trap 50 (FIG. 1a). Optionally, a chemical trap 40 can also be provided, which may contain soda lime, NaOH on a silica substrate, Carbosorb® or the like. The chemical trap 40 may allow removal of carbon dioxide from the analyte gases when it is only desired to look at nitrogen isotope ratios. The moisture trap 50 usually contains Magnesium perchlorate to trap any water generated during the combustion process. Depending upon the nature of the reagents, the chemical trap 40 and moisture trap 50 may be placed in the reverse order to that shown in FIG. 1a. 
The dried gaseous output is introduced into a separation column 60 that serves to separate the output into its constituent atoms, molecules or compounds, e.g. carbon dioxide and nitrogen or carbon dioxide, nitrogen and sulphur dioxide. The separation column 60 may be a packed column for gas chromatography (GC) having a constant temperature when the dried gaseous output flows through the GC column, the GC column being heated by a resistance heater 62 surrounding the GC column 60. The resistance heater 62 is controlled by a heater controller 68 to keep the temperature of the GC column constant. This heater controller 68 is triggered to start the heating by the system controller 100. The arrangement of FIG. 1a shows a separation column 60 in the form of a GC column, with the moisture trap 50 arranged before the separation column 60 as described above.
Once the analyte gas has been separated into its combustion components based on their interaction with the separation column 60, they are conveyed through a thermal conductivity detector (TCD) 80, which forms the basis of weight % determinations. Detection by the TCD 80 is non-destructive. Therefore, after detection, the gas can be conveyed to an isotope ratio mass spectrometer, via an interface capable of diluting the gas if required (not shown in FIG. 1a), for simultaneous measurement, in particular of δ13C, δ15N and/or δ34S values.
Before or after the measurement of an isotope ratio by IRMS, or in parallel with the measurement of an isotope ratio by IRMS, a reference gas of the investigated isotope ratio can be supplied to the IRMS in order to allow a reference measurement to be carried out. The reference gas may be supplied via a gas supply pipe 70 and is under the control of a reference gas supply controller 72 The reference gas supply controller 72 is connected with a bottle 73 of N2, a bottle 74 of CO2 and a bottle 75 of SO2. The measured isotopic ratio is an average for the whole sample. EA-IRMS is particularly suited to non-volatile substances such as soils, sediments, plants, foods, drugs, amino and fatty acids, and many more. Although an average isotope ratio value for the whole sample is obtained, nevertheless analysis of very small samples is possible.
The separation column 60 could also be a thermal desorption unit for gas separation. In such a desorption unit, the thermal desorption temperature is varied as described in EP-A-1 831 680. If the separation column is instead a thermal desorption unit, the moisture trap 50 may be also arranged after the separation column 60.
The thermal desorption unit uses the principle of thermal desorption. Gases emerging from the reduction oven are supplied to the desorption unit. The entire mixture of components of the gas is adsorbed by the adsorbing material of the thermal desorption unit. This adsorption takes place at temperatures between room temperature and 50 degrees Celsius, in systems having a single thermal desorption unit (systems having multiple thermal desorption units are also known, and in these, the lower end of the temperature range may be above room temperature).
The whole gas is stored and can be concentrated by the adsorbing material. Separation of the components of the gas takes place based on different desorption temperatures. Thus, the thermal desorption unit has to be heated to various temperatures to supply specific components of the gas to the EA-IRMS. Due to the control of the desorption of specific elements by the heating temperature it is possible to control the time of the supply of specific component of the gas to the EA-IRMS and the time between the supply of two specific component of the gas to EA-IRMS to be analysed.
GC-IRMS, by contrast, permits separation of the sample prior to isotope ratio analysis. This in turn permits isotopic analysis of complex mixtures by a specific isotope analysis of each chemical substance contained in the mixture, which can reveal additional information not normally available using EA-IRMS, as well as better discrimination. FIG. 1b shows a typical arrangement of a GC-IRMS system, again in highly schematic form. Components common to FIGS. 1a and 1b are labelled with like reference numbers.
Liquid samples (not shown) are provided in small vials (not shown) and loaded into an autosampler 10. The samples are injected by the autosampler 10 into a gas chromatograph (GC) column 60 e.g. by a syringe system (not shown). The gas chromatograph (GC) 60 can be heated in a GC oven 110 under the control of a system controller 100 to improve the separation of the chemical substances contained in the mixture of the investigated sample. The GC oven 110 includes a vent 120. The sample elutes from the column of the GC 60 into an oxidation chamber 20, such as a non-porous alumina tube, usually mounted on the side of the GC oven 100. The eluents from the GC 60 are combusted at elevated temperatures e.g. into NOx, CO2, and/or H2O. As with the EA-IRMS of FIG. 1a, to measure e.g. δ13C, the resulting products are carried in a stream of dry Helium through a reduction oven 30 that converts the nitrous oxides into N2 and removes any excess O2. Water (which is a byproduct of the combustion) is removed using a counterflow of dry helium in a dryer 130, and the dried gaseous output may be introduced into a Thermal Conductivity Detector (TCD) 80.
The gases exiting the TCD 80 are carried into an IRMS (again not shown in FIG. 1b) using CO2 from a reference CO2 supply 70 that is introduced at an open split.
As with the arrangement of FIG. 1a, various components in FIG. 1b are under the control of the system controller 100. The system controller 100 controls the autosampler 10 as it supplies a sample to the combustion oven 20, triggers the supply of the purge gas to the autosampler 10 via the first gas supply control 14, and triggers the supply of the carrier gas flow and the (optional) combustion-assisting oxygen pulse via the second gas supply control 15. The system controller also sets the set-points of the temperature of the combustion oven 20 and the temperature of the reduction oven 30. Finally the system controller 100 controls the temperature of the GC oven 110 which heats the GC column 60. As noted above, EA-IRMS and GC-IRMS are complementary techniques. GC-IRMS allows a specific analysis of each chemical substance contained in a sample, e.g. an organic matter sample (for example, individual amino acids in a protein), but requires that any compound constituting the sample mixture can be made sufficiently volatile and thermally stable to permit initial elution in a GC. It also allows analysis of very small sample quantities (nanogram to picogram range; the typical sample weight in an EA-IRMS experiment is in the milligram to microgram range). The main drawbacks of GC-IRMS are the considerably longer analysis time (typically hours rather than minutes as with EA-IRMS), loss of sample integrity during sample preparation, cost and user complexity. Due to the separation of the chemical substances of the sample by the GC column, the different atoms, molecules and/or compounds of each separated chemical substance are supplied to the mass analyser simultaneously during the GC-IRMS measurement. The different atoms, molecules and/or compounds such as N2, CO2 and SO2 of each chemical substance are very difficult to resolve in such systems. Therefore the measurement results of GC-IRMS are much more complex, or on the other hand different isotope ratios have to be measured one after the other which is very time consuming.
The present invention relates to EA-IRMS, which allows isotopic analysis of the whole samples. One of the key benefits of EA-IRMS is the relatively short time needed for sample analysis. In recent years, simultaneous δ13C, δ15N and δ34S measurements have become a more common approach in EA-IRMS across all application fields. This is because of the ability to produce accurate and precise data from one sample drop, thus increasing system productivity and reducing sample analysis costs. However, such simultaneous measurements in EA-IRMS present a number of challenges. FIG. 2 illustrates a chromatogram for simultaneous δ13C, δ15N and δ34S analysis of sulfanilamide using GC separation at a constant temperature (isothermal) in an EA-IRMS experiment such as that described in connection with FIG. 1a above.
Carbon dioxide, nitrogen and sulfur dioxide molecules generate peaks in the chromatogram of FIG. 2. These molecules are contained in the dried gaseous output of the moisture trap 50 after a sample has been introduced into the sample introduction system shown in FIG. 1a. To the left of FIG. 2, mass peaks of N2 molecules (having isotopic masses 28 u (peak 128) and 29 u (peak 129)) may be observed. The mass peaks of CO2 molecules having isotopic masses 44 u (peak 244), 45 u (peak 245) and 46 u (peak 246) are also visible in FIG. 2. Mass peaks of SO2 molecules having isotopic masses 64 u (peak 364) and 66 u (peak 366) may be seen towards the right of FIG. 2. To determine the isotope ratios δ13C, δ15N and δ34S, reference gases are supplied to the IRMS via the gas supply 70 in parallel with the measurement of the molecules originating from the investigated sample. Those peaks in the chromatogram arising from reference gases are labelled with the same reference number as the corresponding sample gas peak, save for the addition of a prefixed “R”. So, for example the peak labelled “128” in FIG. 2 represents the mass peak of N2 molecules having the isotopic mass 28 u, and which originate from the investigated sample. The peak of N2 molecules having the isotopic mass 28 and which are derived from the N2 reference gas is labelled with the reference number “R128”.
The chromatogram of FIG. 2 exhibits relatively poor N2 and CO2 separation (less than 10 seconds, with some loss of N2 peak tail), a high peak width for SO2 (greater than 100 seconds) and long retention time of SO2, which is the time the SO2 need to pass the GC column, resulting in a total analysis time of in excess of 15 minutes, although this time can often be even longer. The GC column is held at a temperature of around 65-80 degrees Celsius, in the experiment in which the FIG. 2 data are derived and is based on a sample of sulfanilamide (C/S ratio of around 2.5). Sufficient baseline separation between N2 and CO2 on the one hand, and CO2 and SO2 is particularly challenging in samples with large CO2 amounts relative to N2 and SO2. For example, analysis of high C/S ratio samples, such as wood (>5000:1), would result in chromatographic separation compromises that would make the analysis using an isothermal technique impossible for δ13C, δ15N and δ34S from a single sample drop, because separation of N2 and CO2 peaks would not be achieved, and the SO2 peak shape for small S concentrations would result in poor reproducibility.
So the CO2 peaks, the N2 peaks and the SO2 peaks show a peak tailing, that is, exhibit peaks that are not very sharp on their tail side. Sharp peaks permit better peak separation, particularly for the N2 peaks and the CO2 peaks, because the tail side of the N2 peaks do not then extend so close to the front side of the CO2 peaks. Also, for peaks that do not exhibit peak tailing, data integration of the peak is better, and determination of the ratio of the various isotopes is improved. This improvement arises particularly from the fact that, for sharp peaks, it is much easier to distinguish the noise measured in an measurement signal of an EA-IRMS, from the signal of a peak. This results in a more accurate data integration of the peak and consequently a more accurate determination of the ratio of the various isotopes measured by the peak. By contrast, peak tailing results in an extension of the measuring time.
It is possible to reduce the analysis time slightly by operating the GC column at a higher constant temperature, in some prior art systems. However, raising the temperature of the GC column results in poorer N2 and CO2 separation. Thus there is a compromise between achieving analytically acceptable data and the time taken to obtain that. To date, an optimal compromise of around 18 minutes per simultaneous NCS analysis, per sample, has been employed.
The alternative, which is to analyse each of δ13C, δ15N and δ34S separately, has its own drawbacks, in terms of an increase in initial sample weighing and preparation time, along with a requirement for at least three times the amount of the sample. In fact, some prior art EA-IRMS systems require repetition of an experiment once or twice before a statistically acceptable accuracy of the data can be achieved. In such cases, attempting to analyse δ13C, δ15N and δ34S separately can in fact result in up to 6 times more analyses than a simultaneous δ13C, δ15N and δ34S analysis. This results in additional costs per analysis, a longer overall sample preparation time, and lower system productivity (that is, a lower throughput of a specific sample).
Various solutions to these problems have been proposed. One solution employs two GC columns, an S column for the SO2, and an NC column for the N2 and CO2 molecules. The dried gaseous output of a moisture trap 50, containing of N2, CO2 and SO2, flows initially into the S column. The gas flow downstream of the S column can be switched by way of a valve. The valve is initially in a first position which directs the gas flow out of the S column directly to the IRMS, in order that it may be analysed thereby. Once the SO2 has passed through the S column, the valve is switched into a second position so that the gas flowing out of the S column is instead directed next to the NC column. The gas flow out of the NC column is then directed to the IRMS to be analysed. Using this arrangement, the sequence of the molecules to be analysed is changed: initially the SO2 peak is measured by the IRMS, and subsequently the N2 and CO2 peaks are measured by the IRMS. Measurement time can be reduced by the use of a shorter column length of the S column, and larger quantities of CO2 can be measured. Overall, however, the measurement time for the method may be increased, because (at least for a part of the analysis period), the gas is required to flow through two columns (the S and the NC columns) before being measured. Moreover, the costs for this arrangement are higher because of the use of two GC columns as well as an additional controlling system for controlling the additional switching valve.
Also the use of a thermal desorption unit as separation column 60 has its disadvantages. A process of continuously flowing gas into the separation column 60 is not employed. Instead, it is necessary initially to adsorb the whole mixture of gases to be analysed, with the separation column 60 at a low temperature. Only then, by controlled elevation of the temperature, are the specific components to be analysed set free (by a process of desorption) and supplied to the EA-IRMS. This process is time consuming and more difficult to control. Also, the accuracy of the measurement suffers, because it is possible that the specific elements to be analysed are not completely adsorbed during the initial phase of analysis, so that they cannot subsequently be desorbed.
The present invention seeks to address these challenges with existing EA-IRMS devices and methods. It is one of the objects of the invention to reduce the measurement time for the elemental analysis system. It is another one of the objects of the invention to improve the distance between the peaks of different atoms, molecules and/or compounds in the measurement results of the elemental analysis device and to achieve a better peak separation. It is still another one of the objects of the invention to improve the peak shape of the detected atoms, molecules and/or compounds by minimising peak tailing and reducing the peak width. It is still another one of the objects of the invention to expand the range of sample types that may be analysed; for example, it is an object to permit analysis of samples having a high C/S value such as wood. It is still another one of the objects of the invention to reduce the experimental costs associated with the elemental analysis system, for example by reducing the amount of the investigated sample that is needed for successful analysis, and/or by reducing the amount of the flow gases that are needed.